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대한화상학회지제 11 권제 1 호 22 Journal of Korean Burn Society Vol. 11, No. 1, 22-26, 2008 ph 변화가섬유아세포증식에미치는영향 김영석ㆍ황용석ㆍ노태석ㆍ유원민ㆍ탁관철 1 연세대학교의과대학영동세브란스병원성형외과학교실, 1 인체조직복원연구소 The Effect of ph on the Fibroblast Growth Young Seok Kim, M.D., Yong Seok Hwang, M.D., Tai Suk Roh, M.D., Ph.D., Won Min Yoo, M.D., Ph.D. and Kwan Chul Tark, M.D., Ph.D., FACS. 1 Department of Plastic & Reconstructive Surgery, Yong Dong Severance Hospital, Yonsei University College of Medicine, 1 Institute for Human Tissue Restoration, Seoul, Korea Purpose: Fibroblasts are exposed to an acidotic or alkalotic environment in a variety of pathological and physiological conditions. However, the effect of ph changes on fibroblast is still largely unknown, and it was evaluated in the present study using thymidylate synthase activity and cell count. Methods: NIH3T3-mouse fibroblasts were used in this study. Fibroblasts were divided into 5 groups in 6-well plate and fibroblasts (2 10 5 cells in each) were grown in 37 o C 5% CO 2, DMEM and 10% FBS culture media for 4 days. Each well was controlled in the condition of either acidosis or alkalosis (ph 6.0, 6.5, 7.0, 7.5, 8.0). To evaluate the effect of acidosis or alkalosis, cell survival rate and thymidylate synthase (TS) catalytic ratio were used. Results: Mouse fibroblasts showed highest survival rate in ph 7.5 (neutral condition) and the survival rate decreased in both high and low ph. But, the survival rate decreased more profoundly in alkalotic condition. TS catalytic activity ratio showed various changes in either low ph or high ph through the time interval. More significant changes of TS catalytic activity ratio were observed in the high ph. Conclusion: These results show that acidosis and alkalosis inhibit fibroblast proliferation and TS catalytic activity shows similar pattern in low ph but paradoxical changes in high ph because of the cell protective mechanism. (Journal of Korean Burn Society 2008;11:22-26) 책임저자 : 유원민, 서울시강남구도곡동 146-92 135-720, 연세대학교의과대학성형외과학교실 Tel: 02-2019-3421, Fax: 02-3463-4914 E-mail: wnmnyoo@yuhs.ac 본연구는연세대학교의과대학 1998 년도교수연구비에의하여이루어졌음 ( 과제번호 : 6-1998-1102). Key Words: Fibroblast, Growth, ph, Acidosis, Alkalosis, Thymidylate synthase 서 창상치유는외과영역에서오래전부터가장기본이되고많이연구되어온분야이다. 창상이란정상적인조직혹은장기의연속성 (continuity) 가깨진상태를의미한다. 창상치유에는각질세포 (keratinocyte), 혈소판 (platelet), 대식세포 (macrophage) 등많은세포들이관여하지만, 그중에서도섬유아세포 (fibroblast) 가중요한역할을하게된다. 섬유아세포는조직손상에의한염증반응에의해수일내에빠른증식을보이게된다 1). 섬유아세포는세포외기질 (extracellular matrix; ECM) 단백질의주된근원이되면육아조직 (granulation) 을형성하는교원질 (collagen) 과섬유결합소 (fibronectin) 를생산하게된다. 또한근섬유아세포 (myofibroblast) 는창상수축을일으켜치유를돕게된다. 이러한섬유아세포의역할에는 TGF-b (transforming growth factor-beta), PDGF (platelet derived growth factor), VEGF (vascular endothelial growth factor), FGF (fibroblast growth factor) 등의싸이토카인 (cytokine) 이작용을하게되는데이중 TGF-b가가장강력한역할을하는것으로알려져있다 2). TGF-b는세포성장및분화, 이동 (migration), 세포외기질생성 (ECM production), 혈관생성 (angiogenesis), 면역반응 (immunity) 등의다양한과정을조절하는것으로알려져있다 3,4). Thymidylate synthase (TS) 는 DNA 합성에필요한 thymidylate 생산에필수적인인자이며곧세포증식이활발할수록 TS activity가증가하는것으로알려져있다. 주로항암제의치료적효과를판정하는데 TS activity가평가되어왔다 5-7). 허혈상태 (ischemia), 당뇨키토산증 (diabetic ketoacidosis), 창상치유 (wound healing), 호흡부전 (respiratory failure), 요독증 (uremia) 등다양한환경들에서산성 ph가나타나게되어서세포증식에영향을미치게된다. 상피세포에있어서는산성화가에서는혈관내피세포 (endothelial 론

김영석등 :ph and Fibroblast Growth 23 cell) Ca 2+ 을증가 (enhance) 시키고, 혈관내피세포표면에내피성부착분자 (intercellular adhesion molecule-1) 발현을유도하는것으로알려져있다 8,9). 이러한산성화는내피세포의기능을억제하고세포자멸 (apoptosis) 를억제하는것으로알려져있다 10). 또한몇가지 cell line에서세포내산성화가세포자멸에대해미치는영향에대해서도보고되어왔다. 저자들은이러한세포내산성도의변화가창상치유에중요한섬유아세포에미치는영향을알아보기위해서다양한 ph 환경속에서섬유아세포의증식및활성에영향을미치는 TGF-b와세포증식의표식자역할을하는 TS의발현정도를통해알아보고자하였다. 대상및방법 1. 섬유아세포배양 (fibroblast culture) 마우스의섬유아세포 (NIH3T3-mouse fibroblast) 를가지고배양을하였다. 세포는사용전까지액화질소에냉동보존하였고실험은 3차례의계대배양을진행한후에진행되었다. 배양방법은 37 o C 5% CO 2 가함유된절대습도공간에서 10% 의 fetal bovine serum (FBS Hyclone, Logan, UT) 와 5% penicillin/streptomycin (Gilson, Rockville, MD) 이포함된 Dulbecco's Modified Eagle Medium (DMEM) (Life Technologies, Inc., Rockville, MD) 에서배양하였다. 2. 다른 ph 환경에서의섬유아세포배양 DMEM과 10% FBS가각 well에포함된 6-well 플레이트 (Corning Costar, Cambridge, MA) 에섬유아세포 2 10 5 개씩을넣고세포외 ph 영향을평가하기위해서각well의배양배지를 ph 6.0, 6.5, 7.0, 7.5, 8.0으로나누어맞추고배양 하였다. 각 ph 조절을위해서는 1N HCl과 1M의 NaOH가사용되었다. 각각의다른 ph하에서 4일간 37 o C 5% CO 2 가함유된절대습도공간에서배양하였다. 3. 세포생존율및 TS catalytic activity 측정각기다른 ph 환경에서배양된마우스의섬유아세포를배양전및배양 1일, 2일, 3일, 4일째에세포생존율및 TS catalytic activity ratio를측정하였다. 세포생존율을보기위해 trypsinisation 시킨후에 trypan blue exclusion을이용하여생존세포수를측정하였다. 생존율은각시간별 ph 7.5의생존세포수를기준으로하여각각의 ph에서의생존세포수의비를백분율로표시하였다. TS activity는 Mirjolet 등 11) 이기술한바대로측정하였다. 그들보고에서는 TS가 [5-3 H]dUMP를 dtmp로전환시키면서발생되는삼중수 (tritiated water) 를이용해그발현도를측정하였다. 각샘플을 37 o C 에서 15분간배양시킨후, 15% 활성차콜 (activated charcoal) 이포함된 4%trichloroacetic acid를 300μL 첨가한다. 5000xg, 10분간원심분리시킨후에상층액 150μL에존재하는 radioactivity를 liquid scintillation counting을이용해서측정하였다. 이렇게측정한 TS activity를바탕으로각시간대별 TS catalytic activity ratio (=TS activity 각 ph/ts activity ph7.5 100(%)) 를구하여비교하였다. 결과 1. ph변화가섬유아세포생존율에미치는영향마우스섬유아세포는 ph 7.5에서가장높은생존율을보였으며, ph가높거나낮은경우모두에서생존율이감소하였다. 섬유아세포생존율은 ph 7보다 ph 8에서보다낮게 Fig. 1. Cell survival and TS catalytic activity ratio at day 1. Fig. 2. Cell survival and TS catalytic activity ratio at day 2.

24 대한화상학회지 Vol. 11, No. 1, 2008 나타났고이는시간이지남에따라큰차이를보이지않았다. ph가낮아질수록점차섬유아세포의생존율은감소하는것으로나타났다 (Fig. 1 4). ph변화가 thymidylate synthase activity에미치는영향 TS catalytic activity는각시간의변화에따라다양하게나타났는데 1일째에는 ph에따른변화가 ph 7.5에서가장높게나타났고 ph 7.0에서는큰차이를보이지않았으며 ph 6.0, 6.5, 8.0에서는 activity가떨어져있는것으로나타났다. 섬유아세포의생존율과비슷한정도로나타났다 (Fig. 1). 하지만 2일째에는 TS activity가 ph 6.5와 8.0에서가장 높고 7.0과 6.0에서가장낮게나타났다 (Fig. 2). 3일째에는 ph 8.0에서오히려 TS activity가증가하는양상을보였으며 ph가낮을수록감소하는양상이나타났다 (Fig. 3). 4일째에는 ph감소할수록생존율이감소하는것은비슷했으나 ph 8.0에서보다 TS activity ratio가증가함을나타났다 (Fig. 4, 5). 고찰세포외 ph의변화는창상치유에큰영향을미치게된다. Fig. 3. Cell survival and TS catalytic activity ratio at day 3. Fig. 4. Cell survival and TS catalytic activity ratio at day 4. Fig. 5. Microscopic findings of cultured fibroblast survival in various ph conditions at day 4 ( 100).

김영석등 :ph and Fibroblast Growth 25 혈관허혈, 당뇨병성발등의환경에서는산성 ph가나타나게되어서창상치유를지연시키고, 만성창상으로이행시키게되는원인이되기도한다. 이러한산성화는내피세포의기능을억제하고세포자멸을억제하는것으로알려져있다 10). 또한몇가지세포줄기 (cell line) 에서세포내산성화가세포자멸에대해미치는영향에대해서도보고되어왔다. 하지만알칼리환경을포함해서섬유아세포를대상으로 ph 변화를연구한보고는아직없었다. 본연구에서나타난바에의하면섬유아세포의증식은중성 ph (7.5) 에서가장최고점 (peak) 을보이는것으로나타났다. 이러한경향은산성혹은알칼리환경에노출된시간에관계없이항상일정하게나타났다. 그리고 ph가낮거나높을수록섬유아세포생존율이감소하고더구나 ph 가높은환경에서더욱그생존율이감소하는것을알수있었다. 즉중성환경을유지시키는것이섬유아세포의생존율을높여보다창상치유를촉진시킬수있다는것을알수있었다. 산성 ph 환경에서는 TGF-b가활성화되는것으로알려져있다 12). 하지만가장강력한섬유아세포자극성장인자인 TGF-b가활성이되지만섬유아세포가감소하는것은아마도본연구가 in vitro 실험이었으므로성장인자가산성화로인하여수용체 (receptor) 에결합하는친화력 (affinity) 가감소하기때문이고그수용체가감소하기때문에세포내로의신호전달이되지않기때문이라고생각된다. 물론 in vivo에서는이러한과정과는다른과정 (pathway) 을통해, 혈관신생화나교원질생산이촉진되어섬유아세포의활성이증가하는것으로여겨진다. 반면 ph 가높은알칼리환경에서는 (ph 8.0) 섬유아세포가 TGF-b의비활성화때문에증식하지못하고생존율이감소한것으로사료된다. 이것은 in vivo에서나타나는현상과비슷한결과로여겨진다. Thymidylate synthase (TS) 는 DNA 합성에필요한 thymidylate 생산에필수적인인자이며곧세포증식이활발할수록 TS activity가증가하는것으로알려져있다. 본연구에서 TS activity ratio는 1일째에는섬유아세포생존율의변화와비슷한양상으로나타났고 2일째에는알칼리환경에서 TS activity가증가한것을제외하고는큰의미를나타내지못했다. 3일째에는다시섬유아세포생존율의변화와비슷한양상을보였으나알칼리환경에서 TS activity가증가하는양상을보였고 4일째에서는 3일째와비슷한양상을보였으나알칼리환경에서보다 TS activity가크게증가하는것으로나타났다. 본연구에서 ph가낮은환경에서는 TS의변화는큰의미를나타내지못했다. 하지만 ph가높은알칼리환경에서는 TS가예측과는달리매우높게나타났는데그것은세포의방어적메커니즘 (protective mechanism) 이알칼리환경에서시작되어서오히려 TS activity가증가하는것으로사료된다. 실제로 TS activity가증가해도 섬유아세포는증식되지않았다. 그이유는앞서 TGF-b와마찬가지로세포내활성은증가되지않았기때문으로사료된다. 본연구는다양한 ph 환경에서섬유모세포의증식및이와관련된 TS activity의변화를처음으로밝혔다는데의의가있다하겠다하지만좀더일관성있는결과를얻기위해서는좀더많은실험이필요할것이며추후에는인간섬유모세포를이용한실험이필요할것으로생각된다. 향후 ph와 TS activity blockage를통한인간섬유모세포의이상증식 ( 예 : 켈로이드 ) 억제에기초를마련하는연구가될것이라사료된다. 결 산성화혹은알칼리환경에노출된섬유아세포는그증식이억제되고, TS catalytic activity는산성환경에서는세포증식과비슷한패턴을보이나알칼리환경에서는오히려증가하는패턴을보이기도하였다. 론 REFERENCES 1) Hewitson TD, Becker GJ. Interstitial myofibroblasts in IgA glomerulonephritis. Am J Nephrol. 1995;15:111-117. 2) Rahimi RA, Leof EB. TGF-b signaling: a tale of two responses. J Cell Biol. 2007;102:593-608. 3) Massague J, Blain SW, Lo RS. TGF-b signaling in growth control, cancer, and heritable disorders. Cell. 2000;103: 295-309. 4) Patterson GI, Padgett RW. TGF-b related pathways. Roles in Caenorhabditis elegans development. Trends Genet. 2000;16: 27-33. 5) Jehn C, Geyer P, Johson L. Control of thimidylate synthase mrna content and gene transcription in an overproducing mouse cell line. Mol Cell Biol. 1985;5:2527-2532. 6) Navalgrund L, Rossana C, Muench A, Johnson L. Cell cycle regulation of thymidylate synthase gene expression on cultured mouse fibroblasts. J Biol Chem. 1980;255:7386-7390. 7) Pestalozzi B, McGinn C, Kinsella T, et al. Increased thymidylate synthase protein levels are principally associated with proliferation but not cell cycle phase in asynchronous human cancer cells. Br J Cancer. 1995;71:1151-1157. 8) Ziegelstein RC, Cheng L, Blank PS, et al. Modulation of calcium homeostasis in cultured rat aortic endothelial cells by intracellular acidification. Am J Physiol. 1993;265:H1424-433. 9) Serrano CV Jr, Fraticelli A, Paniccia R, et al. ph dependence of neutrophil-endothelial cell adhesion and adhesion molecule expression. Am J Physiol. 1996;271:C962-970. 10) D'arcangelo D, Facchiano F, Barlucchi LM, et al. Acidosis

26 대한화상학회지 Vol. 11, No. 1, 2008 inhibits endothelial cell apoptosis and function and induces basic fibroblast growth factor and vascular endothelial growth factor expression. Circ Res. 2000;86:312-318. 11) Mirjolet JF, Barberi-Heyob M, Merlin JL, et al. Thymidylate synthase expression and activity: relation to S-phase parameters and 5-fluorouracil sensitivity. Br J Cancer. 1998;78:62-68. 12) Tovbin D, Franch HA, Alpern RJ, Preisig PA. Media acidification inhibits TGF beta-mediated growth suppression in cultured rabbit proximal tubule cells. Proc Assoc Am Physicians. 1997;109:572-579.